Technology is an amazing thing--especially technology that allows veterinarians to produce live foals from horses with very poor fertility and even from those that have died. A number of assisted reproductive technologies (ART) for horses have been optimized for commercial use, and veterinarians discussed these in detail during the 2010 American Association of Equine Practitioners convention, held Dec. 4-8 in Baltimore, Md.

"For the equine practitioner, application of ART can range from simply sending ovaries to a laboratory after a mare's untimely death to setting up complete oocyte and embryo culture systems in a practice," began Katrin Hinrichs, DVM, PhD, Dipl. ACT, professor of Veterinary Physiology and Pharmacology and Patsy Link Chair in Mare Reproductive Studies at Texas A&M University. She discussed several ART methods and techniques as follows.

Postmortem Shipment of Ovaries

If a mare dies but the owner still would like foals from her, her ovaries can be harvested immediately and oocytes (egg cells) from them can be matured in the laboratory, then placed in a recipient mare for fertilization and gestation (more on oocyte transfer in a moment). Alternatively, those matured oocytes can be fertilized in the lab (using intracytoplasmic sperm injection, ICSI, or injecting a sperm cell directly into the egg; more on this in a moment). The fertilized eggs are cultured in the lab and resulting embryos can be placed in recipient mares for gestation.

The best success rates with this method are achieved when oocytes are recovered less than eight hours after the mare's death, said Hinrichs. She added that ovaries recovered from mares following barbiturate overdose (customarily used for euthanasia) and those recovered from live mares under general anesthesia had all yielded live foals, and that these ovaries should be kept at body temperature for short trips of less than two hours, or at room temperature for longer trips. She also recommended the use of high-quality semen for these oocyte transfer situations, given the limited oocytes available.

Historically, researchers have reported success rates of 32% when they've transferred harvested oocytesinto recipients for fertilization,, noted Hinrichs. They achieved rates of 63% when oocytes were fertilized via ICSI and embryos were placed in recipients.

It's also possible to harvest the oocytes from ovaries and ship those; Hinrichs described the harvesting and handling procedures that she and her colleagues have found successful in their laboratory at Texas A&M.

Oocyte Recovery from Live Mares

Sometimes a mare's oocytes are fertile, but she can't maintain a pregnancy because of uterine problems. For these mares, veterinarians can surgically harvest mature or immature oocytes for fertilization and/or transfer into recipient mares. Each oocyte type has its advantages. It's easier to harvest mature oocytes from pre-ovulatory follicles, said Hinrichs, but these oocytes are very sensitive to temperature and other environmental influences; thus, shipping and handling can compromise their viability. In contrast, immature follicles are more resource-intensive to harvest and yields can be low, but they're less fragile.

Oocyte Transfer

This method is "currently the most effective method for getting a foal from a mare's isolated oocyte," said Hinrichs. Some studies have reported pregnancy rates of up to 83% with this procedure, in which a recovered oocyte is transferred into the oviduct of a recipient mare, then the recipient is inseminated. These success rates were achieved in healthy, fertile horses; she estimated that in practice, when subfertile horses are used, success rates might be around 35% per cycle.

In Vitro Fertilization

In vitro fertilization (IVF) in humans, where a sperm and egg are put together in culture and allowed to fertilize, doesn't work well in horses because the sperm don't usually penetrate the egg's outer covering. However, Hinrichs described one IVF study in which researchers found that inducing hyperactive sperm movement resulted in a 60% fertilization rate, thus "opening up an exciting area for further investigation in equine IVF," commented Hinrichs. Although embryos have not developed after fertilization with this strategy, researchers continue to work on it.

"Establishment of effective methods for standard IVF would allow use of this ART in many equine practices, because it alleviates the need for micromanipulation equipment and associated expertise," said Hinrichs.

Intracytoplasmic Sperm Injection (ICSI)

As mentioned earlier, this technique involves injecting a single sperm into an oocyte in the laboratory, and is thus useful when fertilizing harvested oocytes and/or when only a limited amount of viable sperm is available for use (such as when there are limited quantities available, or the stallion is subfertile or deceased). Fresh and frozen semen can be used.

Typically, 20 to 35% of oocytes subjected to ICSI produce embryos that can be transferred. Pregnancy rates after transfer of embryos produced with this procedure have increased with improved embryo culture conditions, and they were as high as 83% in one report. Although no published reports exist on the health of foals produced with ICSI, Hinrichs reported that Texas A&M-produced ICSI foals were all normal and healthy from birth.

Cloning (Nuclear Transfer)

Only four laboratories have reported the birth of foals produced by cloning of cells from adult horses. Cloning involves harvesting a small piece of subcutaneous tissue from a donor animal using sterile techniques, removing the nucleus (genetic material) from one of the cells, and transferring the material into an oocyte that has had its genetic material removed. Pregnancy rates approach that of normal breeding methods, although about half those embryos are lost throughout gestation, said Hinrichs. She estimated that in her lab about 30% of transferred cloned embryos produce live foals, which is "very efficient compared to other species."

While many think of cloning as a way to reproduce a proven performance horse, Hinrichs reported that cloned foals have a higher incidence of health problems such as maladjustment and contracted tendons just after birth (which could affect adult athletic ability), and as such they might be best used as breeding animals rather than performance ones.

Another factor in cloning is that although some breed and competitive associations will accept cloned foals as competitors, most breed registries won't register cloned foals largely because of a mitochondria issue. Mitochondria are tiny organelles within cells that synthesize energy to carry out the cell's functions, explained Hinrichs, and they come from the oocyte that receives the donor's genetic material. Those mitochondria could be from any breed of horse, and variation in mitochondria from the donor's mitochondria could theoretically offer variations in growth, stamina, and possibly other traits compared to the original animal. "No real information is available on the effect of mitochondrial origin in the horse," Hinrichs added.

This is an issue for cloned mares, but not for stallions, because mares will pass on their mitochondria via their oocytes, whereas stallions will not. Texas A&M researchers are currently studying cloning using oocytes from mares of the same maternal line as the genetic material donor, reported Hinrichs.

"That way you'd get a foal with the same mitochondria as the original horse," she explained.

Embryo Biopsy for Genetic Testing

An embryo before biopsy (left) and after (right).

If an embryo is being handled in the lab, why not test it for genetic diseases (such as hyperkalemic periodic paralysis, HYPP, or hereditary regional dermal asthenia, HERDA) before putting it in a recipient mare that will carry it to term? Hinrichs reported that Texas A&M researchers have recently published a description of a technique whereby embryos are biopsied for testing, and the embryo recipients still have a normal pregnancy rate of 83%. Foals born from biopsied embryos were normal, she added.

"This technique should allow horse breeders to avoid the production of individuals affected with these devastating genetic diseases," she said. "We anticipate that this technique will allow for the eventual elimination of these diseases from the industry altogether."

So far, the researchers have had about a 75% success rate in detecting genetic disease with this method, which is technically challenging to perform on just a few cells. "This is not yet clinically applicable; 75% accuracy is not accurate enough yet for me," Hinrichs commented.

Embryos might be implanted before test results are complete, but the test results might lead a producer to terminate a pregnancy if the foal is diseased. Or, alternatively, the embryo can be preserved using the last procedure Hinrichs discussed--vitrification, or freezing for storage.

Embryo Freezing (Vitrification)

Although veterinarians have been freezing small embryos successfully for many years, their efforts at freezing larger embryos have not fared as well, Hinrichs reported. However, in the course of researching embryo biopsy, Texas A&M University researchers discovered that large biopsied embryos had good survival rates after freezing, and their implantation resulted in pregnancy rates greater than 50%.

"It is possible that blastocyst collapse (collapse of the rounded embryo's shape when cells were removed and less fluid was contained inside) may allow even standard cryopreservation procedures to be performed; this is a currently developing area," said Hinrichs.

"The year 2010 brings to an end a decade of amazing progress in equine ART," she concluded. "The next decade should show a notable increase in efficiency and accessibility of many equine ART procedures."

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